Endogenous proteases make up a large class of enzymes that are necessary in the normal turnover of proteins and peptides in living organisms. Serine proteases represent an important class of proteases characterized by an essential serine residue in the catalytic site of the enzyme. Physiologically important examples of serine proteases include trypsin, chymotrypsin, tissue kallikrein, cathepsin G, and the elastases such as leukocyte, pancreatic and human neutrophil elastases.
Protease systems are normally highly regulated to properly balance the degradative activities of cells. This regulation is in part accomplished by coordinating the synthesis of proteases with the synthesis of endogenous protease inhibitors. When the normal regulatory mechanisms become unbalanced, protease over-activity can result in devastating disease states. Elevated or unregulated protease activity has been implicated in the pathology of diseases such as respiratory distress syndrome, septic shock, multiple organ failure, emphyzema, myocardial ischemia reperfusion injury, dermatitis, cystic fibrosis, chronic bronchitis, arteriosclerosis, Alzheimer""s disease, corneal ulcers, rheumatoid arthritis and acute pancreatitis, among others.
In addition to the endogenous inhibitors and their physiological roles, natural and synthetic protease inhibitors find valuable utility as research tools for biochemists and medical researchers studying protease systems, cell biology and a variety of diseases.
Despite the importance of the serine proteases and their implication in numerous disease states, effective therapies directed toward inhibition of serine proteases have been hampered by a need for greater specificity in targeted inhibition of individual serine proteases for a given disease state. Furthermore, many inhibitors to date have lacked sufficient stability under physiological conditions to be considered for therapeutic use.
It has been known that peptidyl sulfonyl fluorides, such as phenylmethylsulfonyl fluoride, can be irreversible inhibitors of serine proteases. They are selective for serine proteases in that they tend to be efficient substrates for cysteine proteases and therefore do not irreversibly inhibit cysteine proteases. However, sulfonyl fluorides are not stable under physiological conditions. They are also general inhibitors of serine proteases and are not selective for individual proteases.
Another drawback of sulfonyl fluoride derivatives has been the lack of facile procedures to produce series of tailored sulfonyl fluoride derivatives. Halosulfonyl moieties are highly reactive and it has previously been difficult to generate them or efficiently use them in synthetic pathways involving peptides due to unwanted side reactions.
Peptidyl chloromethane derivatives have also proven effective inhibitors of serine proteases, but are too reactive as general alkylating agents to be useful in vivo. These compounds tend to indiscriminately alkylate most available nucleophiles and are therefore only useful in simple, well defined research systems.
A new family of peptidyl derivatives has been discovered that overcomes these and other drawbacks to the presently known serine protease inhibitors. It has now been surprisingly found that the peptidyl sulfonyl imidazolide compounds of the present invention provide physiologically stable compounds useful as selective, irreversible inhibitors of serine proteases.
It is an object of the present invention to provide serine protease inhibitors for research and therapeutic uses. It is also an object of the invention to provide a family of inhibitors whose members may be tailored to be inhibitors of serine proteases generally, or to be selective inhibitors for specific proteases or subsets of proteases within the serine protease family. Yet another object of the present invention is to provide serine protease inhibitors that are stable under physiological conditions.
Another object of the present invention is to provide a facile synthetic route to a series of sulfonyl derivatives, particularly to xcex1-amino or peptidyl compounds characterized by the conversion of at least one carboxy group to an imidazolesulfonylmethyl group.
Yet another object of the present invention is to provide therapeutic compositions comprising peptidyl sulfonyl imidazolides as serine protease inhibitors useful in the treatment of disease states characterized by an over-activity of one or more serine proteases. Another aspect of the present invention is to provide methods of use of such therapeutic compositions to treat such disease states.
These and other objects and aspects of the present invention are addressed by the new imidazolesulfonylmethyl peptide derivatives described herein and the novel synthetic methods used for their manufacture.
The serine protease inhibitors of the present invention are xcex1-amino or peptidyl compounds having a C-terminal imidazolesulfonylmethyl group. In one embodiment, the inhibitor compound is characterized by the formulas 
wherein Y is a group capable of interacting with the substrate recognition site of a serine protease, R1 is hydrogen or branched or unbranched, substituted or unsubstituted C1-C12 alkyl, aryl, alkaryl, aralkyl or acyl, and R2 is branched or unbranched C1-C6 alkyl. In a preferred embodiment, Y comprises an xcex1-amino group or an amino acid chain of from 2 to 12 amino acid residues. It is preferred that the xcex1-amino group or sequence of amino acid residues in the inhibitor compound is chosen to selectively bind to the substrate recognition site of the protease to be inhibited.
Y may also be of the formula 
wherein R3 is hydrogen or an N-terminal blocking group, A is an amino acid chain of from 0-11 amino acid residues, R4 is an amino acid side chain, b=0 or 1, and c is a covalent bond when b=0 and c not a covalent bond when b=1.
When Y is an amino acid chain, each pair of amino acid residues may be linked to one another by amide bonds or by any other linkage that mimics an amide bond in a peptide. Such alternative linkages may include vinyl, ether, ketomethylene, methylketo, methylamine, aminomethyl, methylene, ethylene, cyclopropylene, thioamide and sulfonamide.
Another aspect of the present invention provides a method of inhibiting a serine protease comprising the step of contacting the protease with a peptidyl sulfonyl imidazolide compound as described above. It is preferred that the protease is contacted with at least a stoiciometric amount of the inhibitor compound and that the compound has a sequence of amino acid residues designed to selectively bind to the substrate recognition site of the protease.
Another aspect of the present invention provides a pharmaceutical composition useful in the treatment of human and animal disease states characterized by an over-activity of one or more serine proteases comprising a pharmaceutically acceptable carrier containing a therapeutically effective amount of a peptidyl protease inhibitor compound as described above. The pharmaceutical composition may be formulated for oral administration as a liquid, tablet, capsule, or enteric encapsulated solid form, or it may be formulated for parenteral or I.V. injection or for administration by infusion.
The present invention also provides for a method of treatment of mammalian disease states characterized by an over-activity of one or more serine proteases comprising administering in a pharmaceutically acceptable carrier, a therapeutically effective amount of a pharmaceutical composition as described above.
Another aspect of the present invention provides for a novel synthetic process for converting a carboxyl group on an organic molecule to a substituted or unsubstituted imidazolesulfonylmethyl group by:
i) reacting the organic molecule with an alkylchloroformate to produce an alkylformate anhydride intermediate;
ii) reducing the alkylformate anhydride intermediate with a borohydride salt to produce an hydroxymethyl intermediate;
iii) reacting the hydroxymethyl intermediate with an alkyl or arylsulfonyl chloride to produce a sulfonate intermediate;
iv) reacting the sulfonate intermediate with cesium alkyl or arylcarbothioic acid to produce a thioic acid S-ester intermediate;
v) reacting the thioic acid S-ester intermediate with Cl2 and water to produce a sulfonyl chloride intermediate; and
vi) reacting the sulfonyl chloride intermediate with substituted or unsubstituted imidazole to produce the imidazolesulfonylmethyl derivative.
It is particularly preferred to conduct step i) in the presence of a tertiary amine or equivalent acid scavenger to coordinate protons generated in the reaction. A preferred molar ratio of tertiary amine or acid scavenger to alkylchloroformate is at least 1.
Likewise, it is particularly preferred to conduct step vi) in the presence of a tertiary amine or equivalent acid scavenger to coordinate protons generated in the reaction. It is particularly convenient to use an excess of imidazole as the scavenger.
In a preferred embodiment of steps v) and vi) of the synthetic process, the molar ratio of water to thioic acid S-ester is at least about 2, the molar ratio of Cl2 to thioic acid S-ester is at least about 3, the molar ratio of imidazole to sulfonyl chloride intermediate is at least about 1, and the molar ratio of acid scavenger to sulfonyl chloride intermediate is at least 1. A preferred method of reacting the Cl2 and water with the thioic acid S-ester is to bubble chlorine gas through a liquid comprising the thioic acid S-ester and water.
In one embodiment of the synthetic method of the present invention, the organic molecule having a carboxy group is an amino acid or an amino acid chain of from 2-12 amino acid residues. One particularly useful carboxy group that may be converted to an imidazolesulfonylmethyl group with the present method is the xcex1-carboxy group of an amino acid or the C-terminal carboxy group of an amino acid chain, though the conversion of other carboxy groups on such molecules may be as facile.
In another embodiment of the present invention, there is provided a process for converting a thioic acid S-ester compound to a sulfonyl derivative compound comprising the steps of:
i) reacting the thioic acid S-ester compound with Cl2 and water to form the corresponding chlorosulfonyl intermediate; and
ii) reacting the chlorosulfonyl intermediate with a nucleophile to form the corresponding sulfonyl derivative compound.
Preferred nucleophiles include imidazole, pyrazole, benzimidazole, benzotriazole, 1,2,3-triazole and 1,2,4-triazole.
It is particularly preferred to conduct step ii) in the presence of a tertiary amine or equivalent acid scavenger to coordinate protons generated in the reaction. It is convenient to use an excess of nucleophile as the scavenger.
In a preferred embodiment, the process is run with a molar ratio of water to thioic acid S-ester of at least about 2, a molar ratio of Cl2 to thioic acid S-ester of at least about 3, a molar ratio of nucleophile to thioic acid S-ester of at least about 1, and the molar ratio of acid scavenger to sulfonyl chloride intermediate of at least about 1. The reaction of the thioic acid S-ester with the Cl2 and water may be conducted by bubbling chlorine gas through a liquid comprising the thioic acid S-ester and water.
Further objects, embodiments, features, benefits and advantages of the present invention will be apparent to one of ordinary skill in the art from the following description of the preferred embodiments of the invention.